This invention is in the field of cancer biology and therapy. Specifically, the invention is directed to methods for altering the differentiated state of a cell by altering syndecan-1 expression. The method allows for the normalization of the growth rate and differentiation state of malignant cells, and is based on the stimulation of syndecan-1 expression in the malignant cells. Re-expression of syndecan-1 in such malignant cells promotes their normal differentiated phenotype and prevents their formation into tumors. This method may also be applied to normal cells to maintain their expression of genes characteristic of the differentiated state, e.g., the method may be used to prevent baldness by maintaining keratin production.
In addition, the invention is directed to transcription regulatory elements associated with the syndecan-1 gene, such as the FGF-inducible Response Element (FiRE) and to the use of these transcription regulatory elements for controlling gene expression. The FiRE can also be used to target expression of a reporter gene to wound sites in vivo.
Cell surface proteoglycans play an important role in the regulation of cell behavior (Ruoslahti et al., Cell 64:867-869 (1991)). Through their covalently bound glycosaminoglycan side chains, such proteoglycans can bind various extracellular effector molecules (Jalkanen, et al., in Receptors for Extracellular Matrix, J. MacDonald and R. Mecham, eds., Academic Press, San Diego, pp. 1-37 (1991)). One central challenge in proteoglycan biology is to understand the biological consequences which result from the binding of different effector molecules to cell surface proteoglycans. It is important to determine the intracellular responses triggered by effector binding and how these responses lead to altered cellular behavior. One way to investigate these matters is to create biological models which are dependent on the expression of specific proteoglycans.
Syndecan-1 is the best characterized cell surface proteoglycan (Saunders et al., J. Cell Biol. 108:1547-1556(1989); Mali et al., J. Biol. Chem. 265:6884-6889 (1990)). It was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan side chains (Rapraeger et al., J. Biol. Chem. 260:11046-11052 (1985)). Recent studies have revealed its expression, not only on epithelial cells but also on differentiating fibroblasts of developing tooth (Thesleff et al., Dev. Biol. 129:565-572 (1988); Vainio et al., J. Cell Biol. 108:1945-1964 (1989)), on endothelial cells of sprouting capillaries (Elenius et al., J. Cell Biol. 114:585-596 (1991)) and on the surface of lymphocyte subpopulations (Sanderson et al., Cell Regul. 1:27-35 (1989)). This suggests that syndecan-1 function can vary from one cell type to another. Syndecan belongs to a family of proteoglycans with conserved plasma membrane and cytoplasmic domains but with dissimilar ectodomains (Mali et al., J. Biol. Chem. 265:6884-6889 (1990)). The conserved structure of syndecans suggests that it could participate in signal transduction through the plasma membrane.
Syndecan-1 binds several extracellular effector molecules but does so in a selective manner. For example, syndecan binds interstitial collagens and fibronectin but does not bind vitronectin or laminin (Koda et al., J. Biol. Chem. 260:8156-8162 (1985)); Saunders et al., J. Cell Biol. 106:423-430 (1988); Elenius et al., J. Biol. Chem. 265:17837-17843 (1990)). Moreover, syndecan-1 isolated from tooth mesenchyme has revealed selective binding to tenascin not observed for syndecan from NMuMG cells (Salmivirta et al., J. Biol. Chem. 266:7733-7739 (1991)). This suggests that variations in syndecan glycosylation alters the binding properties of syndecan. Polymorphism of syndecan-1 glycosylation has also been observed in simple and stratified epithelia (Sanderson et al., Proc. Natl. Acad Sci. USA 85:9562-9566 (1988)); but whether these changes also reflect altered ligand recognition by syndecan remains unknown. Syndecan-1 also binds growth factors, such as basic fibroblast growth factor (Kiefer et al., Proc. Natl. Acad. Sci. USA 87:6985-6989 (1990); Elenius et al., J. Biol. Chem. 267:6435-6441 (1992)).
Fibroblast growth factors (FGFs) are a family of heparin-binding peptides comprising 9 known members. Basic fibroblast growth factor (FGF-2 or bFGF) is synthesized by, and acts on various cell types and tissues. In vitro, it is a strong mitogen for cells of mesodermal origin, can modulate cell motility and differentiation, is a potent angiogenic factor, and potentiates neovascularization in vivo (Burgess and Magiac, Ann. Rev. Biochem. 58:575-606 (1989); Mason, Cell 78:547-552 (1994)). Keratinocyte growth factor (FGF-7 or KGF) is produced solely by cells of mesodermal origin. FGF-7 is proliferative for various epithelial cells (Basilico and Moscatelli, Adv. Cancer Res. 59:115-65 (1992); Rubin et al., Cell Biol. Int. 19:399-411 (1995)), and is also an important mediator of hair follicle growth and differentiation (Danilenko et al., Am. J. Pathol. 147:145-54 (1995); Guo et al., Genes and Develop. 10:165-75 (1996)). Both FGF-2 and FGF-7 are involved in wound healing, where they act as both autocrinic and paracrinic factors. In wounded skin FGF-2 is found in fibroblasts and endothelial cells. This growth factor stimulates proliferation of most cell types involved in wound healing, e.g., keratinocytes, fibroblasts, and vascular and capillary endothelial cells (Bennett et al., Am. J. Surg. 165:728-737 (1993)). FGF-7, which is synthesized only by fibroblasts and is induced during wound healing (Wemer et al., Science 266:819-22 (1994); Werner et al., Proc. Natl. Acad. Sci. 89:6898-6900 (1992), and acts as a paracrinic factor on keratinocytes, inducing their proliferation and migration (Bennett et al., supra). FGF-7 is important for normal wound reepithelialization (Werner et al. (1994), supra). However, recent data with FGF-7 knockout mice indicate that KGF may not be required for normal wound healing (Guo et al., Genes and Develop. 10:165-175 (1996).
Growth factors are involved in the initiation, control, and termination processes of wound healing in an autocrinic and paracrinic manner. FGF-2 is produced by fibroblasts and is also found in association with extracellular matrix and basement membranes where it can be released by proteolytic activity. FGF-2 enhances the accumulation and proliferation of fibroblasts, keratinocytes, endothelial cells, and macrophages. In animal models it induces neovascularization, cell migration, and granulation tissue formation. It has been shown to accelerate wound healing in several different situations, e.g., incisions, burns, and diabetic wounds (See Bennett and Schulz, Am. J. Surg. 165: 728-737 (1993)).
Several AP-1 regulated genes are expressed during wound healing. Fos is rapidly activated on the wound healing edge (Martin and Nobes, Mech. Dev. 38: 209-215 (1992)). Jun may also be activated during wounding or wounding induced tumorigenesis (Marshall et al., Virology 188(1): 373-379 (1992). Cancerous cells are also known to be able to activate the AP-1 complex, and c-fos is required for malignant tumor progression (Saez et al., Cell 82:721-732 (1995)).
Yayon and coworkers (Yayon et al., Cell 64:841-848 (1991)) and Rapraeger and coworkers (Rapraeger et al., Science 252:1705-1708 (1991)) have shown that heparin-like molecules are required for the binding of FGF-2 to its high affinity receptor, indicating that syndecan-like molecules can also modulate the growth factor response. It has been observed that heparin is required for oligomerization of FGF-1 molecules leading to FGFR dimerization or further oligomerization and further signaling (Spivak-Kroizman et al., Cell 79:1015-24 (1994); Ornitz et al., Mol. Cell Biol. 12:240-47 (1992)). Several mechanisms, for both negative and positive regulation for FGF action by proteoglycans have been postulated (Schlessinger et al., Cell 83:357-360 (1995)). Syndecan-1 (Saunders et al., J. Cell Biol. 107:1199-1205 (1989)), can simultaneously bind FGF-2 and extracellular matrix molecules and this complex is able to promote DNA synthesis in 3T3 cells (Salmivirta et al., J. Biol. Chem. 267:17606-17610 (1992)).
However, it has also been reported that different heparin sequences can either activate or inhibit FGF-2 function (Guimond et al., J. Biol. Chem. 268:23906-23914 (1993)) and that the composition and length of syndecan side chains vary in a cell and tissue dependent manner (Sanderson and Bemfield, Proc. Natl. Acad. Sci. USA 85:9562-9566 (1989)); Rapraeger, J. Cell Biol. 109:2509-2518 (1989)); Salmivirta et al., J. Biol. Chem. 267:17606-17610 (1991)). Negative regulation of FGF action by syndecan-1 has also been reported (Mali et al., J. Biol. Chem. 268:24251-24258 (1993)); Aviezer et al., J. Biol. Chem. 269:114-121 (1994)), which may be due to the glycosaminoglycan side chain modification or different stochiometric ratios of FGFR and its co-receptor.
FGF-2 induces the transcription of number of genes encoding transcription factors, components of cytoskeleton, and ribosomal components (Burgess and Magiac, supra). For various growth factors and cytokines the inducible downtream transcriptional mechanisms are well characterized.
Several growth factors can elicit immediate early responses after receptor activation. For example, epidermal growth factor (EGF) and platelet derived growth factor (PDGF) induce, via the ras/ERK pathway, the ternary complex factor. The ternary complex factor acts together with the serum response factor to activate the serum response element (SRE) (Hill and Treisman, EMBO J. 14:5037-5047 (1995)). The cAMP response element (CRE) bound by CRE binding protein (CREB) homodimer, or as heterodimers in association with members of the ATF family, are also under the influence of growth factors, e.g., EGF, PDGF, and FGF-2. EGF and PDGF, with various cytokines, are also able to induce activation of signal transducers and activators of transcription (STAT), which are activated by Janus Kinases (JAKs) and act on the sis-inducible element (SIE) or the interferon stimulated response element (IRSE) (Hill and Treisman, Cell 80:199-211 (1995); Karin, Cur. Op. Cell Biol. 6:415-424 (1994)). Transforming growth factor alpha (TGFxcex1) can activate nFkB.
FGF-2 and -7 both signal by binding to different cell surface tyrosine kinase receptors. However, no clear response element has been previously described for FGF and the signal receiving transcription factors remain to be discovered. FGF-2 can also be localized to the nucleus, where it is supposed to activate transcription (Nakiniski et al., Proc. Natl. Acad. Sci USA 89:5216-5220 (1992); Bouche et al., Proc. Natl. Acad. Sci. USA 84:6770-6774 (1987)) as does FGF-1 (Wiedloche et al., Cell 76:1039-1051 (1994)). Accumulating evidence indicates that FGFs activate the MEF-MAP kinase pathway downstream from FGFR activation, via ras and raf (Umbhauser et al., Nature 376:58-62 (1995)). However, other signal transduction pathways also might contribute to FGF signaling, including a phospholipid-C driven pathway (Mason, Cell 78:547-552 (1994)).
Synergistic action of FGF-2 and cAMP by c-Jun and ATF-3 transcription factors at CRE has also been reported (Tan et al., Mol. Cell Biol. 14:7546-7556 (1994)). However, FGF was a poor activator of transcription without cAMP, indicating that besides FGF this type of FGF induced gene activation requires other extracellular signaling molecules. The SER of a-actin promoter, but not the fos promoter, has been proposed to be another target for FGF-2 induced gene activation in a cell culture model of cardiac myocytes (Parker et al., J. Biol. Chem. 267: 3343-3350 (1992)). For FGF induced negative regulation, one established gene element is the E-box motif, which binds myogenic basic helix-loop-helix (bHLH) transcription factors known to be inhibited by FGF-2. Proteins of the MyoD family: MyoD, myogenin, Myf5, and MRF, bind E-box elements in regulatory regions of various myogenic genes and are responsible for the terminal differentiation of myoblasts to myocytes (Edmondson and Olson, J. Biol. Chem. 268:755-758 (1993)).
The FGF-2 signaling effect seems to be dependent on its molecular weight. Different forms of FGF-2 are created in cells by alternative splicing. The low molecular weight form can induce cell migration, but does not have the mitogenic or proliferative potential of the high molecular forms (Bikfalvi et al., J. Cell Biol. 129:233-243 (1995)). The different molecular forms thus seem to use different signaling pathways.
The fact that cell surface proteoglycans can bind both growth factors and matrix components suggests that proteoglycans play a role in regulating, both temporally (timing of expression) and spatially (precise localization), growth promotion by immobilizing effector molecules to the vicinity of cell-matrix interactions. This is supported by the pattern of syndecan-1 expression during development which follows morphogenetic, rather than histological, patterns (Thesleff et al., Dev. Biol. 129:565-572 (1988); Vainio et al., J. Cell Biol. 108:1945-1954(1989); and Vainio et al., Dev. Biol. 134:382-391 (1989)), and by the observation that syndecan expression is localized to sites of active proliferation (Elenius et al., J. Cell Biol. 114:585-596 (1991) and Vainio et al., Dev. Biol. 147:322-333 (1991)).
In simple epithelium, syndecan-1 is polarized to baso-lateral surfaces where it co-localizes with actin rich cytofilaments (Rapraeger et al., J. Cell Biol. 103:3683-2696 (1986)). Upon rounding, syndecan-1 is shed from the cell surface by proteolytic cleavage of the core protein at the cell surface, a process which separates the matrix binding ectodomain from the membrane domain (Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). In this way, syndecan-1 has been proposed to be involved in the maintenance of epithelial morphology. When mouse mammary tumor cells (S115) are induced to change their morphology from an epithelial to a more fibroblastic or fusiform phenotype, syndecan-1 expression is lost (Leppxc3xa4 et al., Cell Regul. 2:1-11 (1991)). This loss has been found to occur in other cell types undergoing transformation (Inki et al., Am. J. Pathol. 139:1333-1340 (1991); Inki et al., Lab. Invest. 66:314-323 (1992)) suggesting that the loss of syndecan-1 expression is a common characteristic of malignant transformation.
The present invention is directed to a method for altering the differentiated state of a host cell by altering its expression of syndecan-1. The invention is also directed to a method for inducing and regulating syndecan-1 expression, especially in cells which exhibit a malignant phenotype, regardless of the origin of transformation.
In another aspect, the invention is directed to a treatment for suppressing tumor growth in a patient in need of such treatment, by the administration of a composition comprised of efficacious amounts of one or more agents that stimulate syndecan-1 synthesis in the tumor cells of such patient.
The invention is also directed to the DNA encoding enhancer and suppressor elements of the syndecan-1 gene and to the use of these elements for regulating heterologous gene expression. The vectors and host cells which incorporate DNA sequences containing the syndecan enhancer or suppressor are also encompassed by the invention. Methods of expressing a standard or heterologous gene comprising activating the syndecan enhancer element are also provided.
In addition, the invention is directed to a method for enhancing syndecan-1 expression in a host cell by activating the enhancer element of the syndecan-1 gene.
The invention is also directed to a method for enhancing syndecan-1 expression in malignant cells, by preventing suppression of syndecan gene transcription.
The invention is also directed to a biochemical method for the inactivation of suppressors of syndecan-1 gene expression in malignant cells.
In another aspect, the invention is directed to a method for stimulating cellular differentiation by enhancing syndecan-l expression in both malignant and normal cells.
The invention is also directed to a method for stimulating cellular proliferation and differentiation, thus promoting tissue regeneration, especially in processes such as wound healing, by enhancing syndecan-1 expression.
This invention also relates to an enhancer element of the syndecan-1 gene having about 280 nucleotides. In addition, this invention relates to nuclear proteins that bind to the syndecan enhancer element causing enhancer activation, to the binding sites for these nuclear proteins on the syndecan enhancer, and to a complex of these nuclear proteins bound to the syndecan enhancer element. In particular embodiments of this invention, this nuclear protein is FIN-1, AP-1, or USF. In more specific embodiments, one or more of these proteins are in an oligomeric form, such as a dimer.
The invention further relates to a method of targeting expression of a gene product to cutaneous wounds using a construct comprising the syndecan enhancer gene operably linked to the gene of interest. Non-human transgenic animals comprising the syndecan enhancer operably linked to the gene of interest inserted into this germ line are also provided.
In another aspect, this invention relates to a method of activating or enhancing syndecan-1 expression in malignant cells by activating the syndecan enhancer element. In specific embodiments of this invention, the enhancer element is activated by FIN-1.
Further features, objects and advantages of the present invention will become more fully apparent to one of ordinary skill in the art from a detailed consideration of the following description of the subject invention when taken together with the accompanying drawings.